Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK

Abstract

The related protein kinases SPAK and OSR1 regulate ion homeostasis in part by phosphorylating cation cotransporter family members. The structure of the kinase domain of OSR1 was solved in the unphosphorylated inactive form, and like some other Ste20 kinases, exhibited a domain-swapped activation loop. To further probe the role of domain swapping in SPAK/OSR1, we have determined the crystal structures of SPAK 63–403 at 3.1 Å and SPAK 63–390 T243D at 2.5 Å resolutions. These structures encompass the kinase domain and different portions of the C-terminal tail, the longer without, and the shorter with an activating point mutation T243D. The structure of the T243D protein reveals significant conformational differences relative to unphosphorylated SPAK and OSR1, but also has some features of an inactive kinase. Both structures are domain-swapped dimers. Sequences involved in domain swapping were identified and mutated to create a SPAK monomeric mutant with kinase activity, indicating that monomeric forms are active. The monomeric mutant is activated by WNK1, but has reduced activity toward its substrate NKCC2, suggesting regulatory roles for domain swapping. The structure of the partially active SPAK T243D is consistent with a multi-stage activation process in which phosphorylation induces a SPAK conformation that requires further remodeling to build the active structure.

Germinal‐center kinase‐like kinase co‐crystal structure reveals a swapped activation loop and C‐terminal extension

“…(A)]. This is consistent with the observation made by Taylor et al . that the presence of a glycine or proline right before the aligned tyrosine after the APE of the activation loop is the critical determinant of whether the STE kinase forms an activation loop swapped dimer or a monomer.…”

“…The HGK structure is an activation loop-swapped dimer, similar to STE-20 kinases such as MST4, SLK (LOSK), OSR1, CHK2, LOK, DAPK3, and SPAK. [19][20][21][22] The MST4 dimer forms an extended beta strand between the "FVGT" sequences of the swapped activation loops, similar to the extended b-strand observed for Map4k4 "TFIGT" sequence. The literature suggests that activation loop swapping allows a single kinase molecule to catalyze phosphorylation to two different subsets of residues (the autophosphorylation sequence of the dimer versus the physiological consensus sequence of the substrate).…”

“…The structure of only one kinase domain of the GCK‐I family has been published to date–Map4k4 or HGK(PDB IDs: 4U43, 4U44, 4U45,4OBO, 4OBP, and others). The HGK structure is an activation loop‐swapped dimer, similar to STE‐20 kinases such as MST4, SLK (LOSK), OSR1, CHK2, LOK, DAPK3, and SPAK . The MST4 dimer forms an extended beta strand between the “FVGT” sequences of the swapped activation loops, similar to the extended β‐strand observed for Map4k4 “TFIGT” sequence.…”

WNK Kinases in Development and Disease

“…This did not occur if wild-type SPAK and the Thr-to-Ala mutant SPAK were introduced as separate monomers, indicating that prior dimerization (experimentally recapitulated by concatamerization) is required to observe the Mo25 effect. The authors proposed that WNK phosphorylation of SPAK allows it to assume a domain swapping-competent conformation, which is further facilitated by Mo25 (Ponce-Coria et al, 2012), consistent with the results of the structural studies of SPAK T243D described above (Taylor et al, 2015). An additional study from the Delpire group, examining mouse and sea urchin OSR1 with or without Mo25, adds additional insights into OSR1 activation mechanisms (Gagnon et al, 2011).…”

“…Based on structural and mutational analysis of MST4, another STE20 kinase that complexes with Mo25, Shi and coworkers proposed that Mo25 may facilitate the trans-autophosphorylation of MST4 dimers in order to fully activate MST4 (Shi et al, 2013). Indeed, the crystal structure of a SPAK mutant in which the WNK target T-loop threonine is mutated to a phospho-mimicking aspartic acid (T243D) demonstrated a partially active conformation, supporting the hypothesis that Mo25 binding allows for full activation of SPAK/OSR1 kinases after partial activation by WNK phosphorylation (Taylor et al, 2015). Further support for the hypothesis that Mo25 facilitates domain swapping in SPAK/OSR1 dimers was provided by an elegant series of experiments by Delpire and colleagues.…”